A Dynamic 6,000-Year Genetic History of Eurasia's Eastern Steppe.

The Eastern Eurasian Steppe was home to historic empires of nomadic pastoralists, including the Xiongnu and the Mongols. However, little is known about the region's population history. Here, we reveal its dynamic genetic history by analyzing new genome-wide data for 214 ancient individuals spanning 6,000 years. We identify a pastoralist expansion into Mongolia ca. 3000 BCE, and by the Late Bronze Age, Mongolian populations were biogeographically structured into three distinct groups, all practicing dairy pastoralism regardless of ancestry. The Xiongnu emerged from the mixing of these populations and those from surrounding regions. By comparison, the Mongols exhibit much higher eastern Eurasian ancestry, resembling present-day Mongolic-speaking populations. Our results illuminate the complex interplay between genetic, sociopolitical, and cultural changes on the Eastern Steppe.

Genomic History of Neolithic to Bronze Age Anatolia, Northern Levant, and Southern Caucasus.

Here, we report genome-wide data analyses from 110 ancient Near Eastern individuals spanning the Late Neolithic to Late Bronze Age, a period characterized by intense interregional interactions for the Near East. We find that 6th millennium BCE populations of North/Central Anatolia and the Southern Caucasus shared mixed ancestry on a genetic cline that formed during the Neolithic between Western Anatolia and regions in today's Southern Caucasus/Zagros. During the Late Chalcolithic and/or the Early Bronze Age, more than half of the Northern Levantine gene pool was replaced, while in the rest of Anatolia and the Southern Caucasus, we document genetic continuity with only transient gene flow. Additionally, we reveal a genetically distinct individual within the Late Bronze Age Northern Levant. Overall, our study uncovers multiple scales of population dynamics through time, from extensive admixture during the Neolithic period to long-distance mobility within the globalized societies of the Late Bronze Age. VIDEO ABSTRACT.

Ancient genomes reveal insights into ritual life at Chichén Itzá.

The ancient city of Chichén Itzá in Yucatán, Mexico, was one of the largest and most influential Maya settlements during the Late and Terminal Classic periods (AD 600-1000) and it remains one of the most intensively studied archaeological sites in Mesoamerica1-4. However, many questions about the social and cultural use of its ceremonial spaces, as well as its population's genetic ties to other Mesoamerican groups, remain unanswered2. Here we present genome-wide data obtained from 64 subadult individuals dating to around AD 500-900 that were found in a subterranean mass burial near the Sacred Cenote (sinkhole) in the ceremonial centre of Chichén Itzá. Genetic analyses showed that all analysed individuals were male and several individuals were closely related, including two pairs of monozygotic twins. Twins feature prominently in Mayan and broader Mesoamerican mythology, where they embody qualities of duality among deities and heroes5, but until now they had not been identified in ancient Mayan mortuary contexts. Genetic comparison to present-day people in the region shows genetic continuity with the ancient inhabitants of Chichén Itzá, except at certain genetic loci related to human immunity, including the human leukocyte antigen complex, suggesting signals of adaptation due to infectious diseases introduced to the region during the colonial period.

The genomic origins of the Bronze Age Tarim Basin mummies.

The identity of the earliest inhabitants of Xinjiang, in the heart of Inner Asia, and the languages that they spoke have long been debated and remain contentious1. Here we present genomic data from 5 individuals dating to around 3000-2800 BC from the Dzungarian Basin and 13 individuals dating to around 2100-1700 BC from the Tarim Basin, representing the earliest yet discovered human remains from North and South Xinjiang, respectively. We find that the Early Bronze Age Dzungarian individuals exhibit a predominantly Afanasievo ancestry with an additional local contribution, and the Early-Middle Bronze Age Tarim individuals contain only a local ancestry. The Tarim individuals from the site of Xiaohe further exhibit strong evidence of milk proteins in their dental calculus, indicating a reliance on dairy pastoralism at the site since its founding. Our results do not support previous hypotheses for the origin of the Tarim mummies, who were argued to be Proto-Tocharian-speaking pastoralists descended from the Afanasievo1,2 or to have originated among the Bactria-Margiana Archaeological Complex3 or Inner Asian Mountain Corridor cultures4. Instead, although Tocharian may have been plausibly introduced to the Dzungarian Basin by Afanasievo migrants during the Early Bronze Age, we find that the earliest Tarim Basin cultures appear to have arisen from a genetically isolated local population that adopted neighbouring pastoralist and agriculturalist practices, which allowed them to settle and thrive along the shifting riverine oases of the Taklamakan Desert.

Reconstruction of ancient microbial genomes from the human gut.

Loss of gut microbial diversity1-6 in industrial populations is associated with chronic diseases7, underscoring the importance of studying our ancestral gut microbiome. However, relatively little is known about the composition of pre-industrial gut microbiomes. Here we performed a large-scale de novo assembly of microbial genomes from palaeofaeces. From eight authenticated human palaeofaeces samples (1,000-2,000 years old) with well-preserved DNA from southwestern USA and Mexico, we reconstructed 498 medium- and high-quality microbial genomes. Among the 181 genomes with the strongest evidence of being ancient and of human gut origin, 39% represent previously undescribed species-level genome bins. Tip dating suggests an approximate diversification timeline for the key human symbiont Methanobrevibacter smithii. In comparison to 789 present-day human gut microbiome samples from eight countries, the palaeofaeces samples are more similar to non-industrialized than industrialized human gut microbiomes. Functional profiling of the palaeofaeces samples reveals a markedly lower abundance of antibiotic-resistance and mucin-degrading genes, as well as enrichment of mobile genetic elements relative to industrial gut microbiomes. This study facilitates the discovery and characterization of previously undescribed gut microorganisms from ancient microbiomes and the investigation of the evolutionary history of the human gut microbiota through genome reconstruction from palaeofaeces.

A primer for ZooMS applications in archaeology.

Collagen peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, also known as zooarchaeology by mass spectrometry (ZooMS), is a rapidly growing analytical technique in the fields of archaeology, ecology, and cultural heritage. Minimally destructive and cost effective, ZooMS enables rapid taxonomic identification of large bone assemblages, cultural heritage objects, and other organic materials of animal origin. As its importance grows as both a research and a conservation tool, it is critical to ensure that its expanding body of users understands its fundamental principles, strengths, and limitations. Here, we outline the basic functionality of ZooMS and provide guidance on interpreting collagen spectra from archaeological bones. We further examine the growing potential of applying ZooMS to nonmammalian assemblages, discuss available options for minimally and nondestructive analyses, and explore the potential for peptide mass fingerprinting to be expanded to noncollagenous proteins. We describe the current limitations of the method regarding accessibility, and we propose solutions for the future. Finally, we review the explosive growth of ZooMS over the past decade and highlight the remarkably diverse applications for which the technique is suited.

Stone Age Yersinia pestis genomes shed light on the early evolution, diversity, and ecology of plague.

The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague's formative years in terms of its early evolution and ecology.

Paleoproteomics.

Paleoproteomics, the study of ancient proteins, is a rapidly growing field at the intersection of molecular biology, paleontology, archaeology, paleoecology, and history. Paleoproteomics research leverages the longevity and diversity of proteins to explore fundamental questions about the past. While its origins predate the characterization of DNA, it was only with the advent of soft ionization mass spectrometry that the study of ancient proteins became truly feasible. Technological gains over the past 20 years have allowed increasing opportunities to better understand preservation, degradation, and recovery of the rich bioarchive of ancient proteins found in the archaeological and paleontological records. Growing from a handful of studies in the 1990s on individual highly abundant ancient proteins, paleoproteomics today is an expanding field with diverse applications ranging from the taxonomic identification of highly fragmented bones and shells and the phylogenetic resolution of extinct species to the exploration of past cuisines from dental calculus and pottery food crusts and the characterization of past diseases. More broadly, these studies have opened new doors in understanding past human-animal interactions, the reconstruction of past environments and environmental changes, the expansion of the hominin fossil record through large scale screening of nondiagnostic bone fragments, and the phylogenetic resolution of the vertebrate fossil record. Even with these advances, much of the ancient proteomic record still remains unexplored. Here we provide an overview of the history of the field, a summary of the major methods and applications currently in use, and a critical evaluation of current challenges. We conclude by looking to the future, for which innovative solutions and emerging technology will play an important role in enabling us to access the still unexplored "dark" proteome, allowing for a fuller understanding of the role ancient proteins can play in the interpretation of the past.

Exotic foods reveal contact between South Asia and the Near East during the second millennium BCE.

Although the key role of long-distance trade in the transformation of cuisines worldwide has been well-documented since at least the Roman era, the prehistory of the Eurasian food trade is less visible. In order to shed light on the transformation of Eastern Mediterranean cuisines during the Bronze Age and Early Iron Age, we analyzed microremains and proteins preserved in the dental calculus of individuals who lived during the second millennium BCE in the Southern Levant. Our results provide clear evidence for the consumption of expected staple foods, such as cereals (Triticeae), sesame (Sesamum), and dates (Phoenix). We additionally report evidence for the consumption of soybean (Glycine), probable banana (Musa), and turmeric (Curcuma), which pushes back the earliest evidence of these foods in the Mediterranean by centuries (turmeric) or even millennia (soybean). We find that, from the early second millennium onwards, at least some people in the Eastern Mediterranean had access to food from distant locations, including South Asia, and such goods were likely consumed as oils, dried fruits, and spices. These insights force us to rethink the complexity and intensity of Indo-Mediterranean trade during the Bronze Age as well as the degree of globalization in early Eastern Mediterranean cuisine.

The evolution and changing ecology of the African hominid oral microbiome.

The oral microbiome plays key roles in human biology, health, and disease, but little is known about the global diversity, variation, or evolution of this microbial community. To better understand the evolution and changing ecology of the human oral microbiome, we analyzed 124 dental biofilm metagenomes from humans, including Neanderthals and Late Pleistocene to present-day modern humans, chimpanzees, and gorillas, as well as New World howler monkeys for comparison. We find that a core microbiome of primarily biofilm structural taxa has been maintained throughout African hominid evolution, and these microbial groups are also shared with howler monkeys, suggesting that they have been important oral members since before the catarrhine-platyrrhine split ca. 40 Mya. However, community structure and individual microbial phylogenies do not closely reflect host relationships, and the dental biofilms of Homo and chimpanzees are distinguished by major taxonomic and functional differences. Reconstructing oral metagenomes from up to 100 thousand years ago, we show that the microbial profiles of both Neanderthals and modern humans are highly similar, sharing functional adaptations in nutrient metabolism. These include an apparent Homo-specific acquisition of salivary amylase-binding capability by oral streptococci, suggesting microbial coadaptation with host diet. We additionally find evidence of shared genetic diversity in the oral bacteria of Neanderthal and Upper Paleolithic modern humans that is not observed in later modern human populations. Differences in the oral microbiomes of African hominids provide insights into human evolution, the ancestral state of the human microbiome, and a temporal framework for understanding microbial health and disease.

Ecology, Not Host Phylogeny, Shapes the Oral Microbiome in Closely Related Species.

Host-associated microbiomes are essential for a multitude of biological processes. Placed at the contact zone between external and internal environments, the little-studied oral microbiome has important roles in host physiology and health. Here, we investigate the roles of host evolutionary relationships and ecology in shaping the oral microbiome in three closely related gorilla subspecies (mountain, Grauer's, and western lowland gorillas) using shotgun metagenomics of 46 museum-preserved dental calculus samples. We find that the oral microbiomes of mountain gorillas are functionally and taxonomically distinct from the other two subspecies, despite close evolutionary relationships and geographic proximity with Grauer's gorillas. Grauer's gorillas show intermediate bacterial taxonomic and functional, and dietary profiles. Altitudinal differences in gorilla subspecies ranges appear to explain these patterns, suggesting a close connection between dental calculus microbiomes and the environment, likely mediated through diet. This is further supported by the presence of gorilla subspecies-specific phyllosphere/rhizosphere taxa in the oral microbiome. Mountain gorillas show a high abundance of nitrate-reducing oral taxa, which may promote adaptation to a high-altitude lifestyle by modulating blood pressure. Our results suggest that ecology, rather than evolutionary relationships and geographic distribution, shape the oral microbiome in these closely related species.

High conservation of the dental plaque microbiome across populations with differing subsistence strategies and levels of market integration.

Industrialization-including urbanization, participation in the global food chain and consumption of heavily processed foods-is thought to drive substantial shifts in the human microbiome. While diet strongly influences stool microbiome composition, the influence of diet on the oral microbiome is largely speculative. Multiple ecologically distinct surfaces in the mouth, each harbouring a unique microbial community, pose a challenge to assessing changes in the oral microbiome in the context of industrialization, as the results depend on the oral site under study. Here, we investigated whether microbial communities of dental plaque, the dense biofilm on non-shedding tooth surfaces, are distinctly different across populations with dissimilar subsistence strategies and degree of industrialized market integration. Using a metagenomic approach, we compared the dental plaque microbiomes of Baka foragers and Nzime subsistence agriculturalists in Cameroon (n = 46) with the dental plaque and calculus microbiomes of highly industrialized populations in North America and Europe (n = 38). We found that differences in microbial taxonomic composition between populations were minimal, with high conservation of abundant microbial taxa and no significant differences in microbial diversity related to dietary practices. Instead, we find that the major source of variation in dental plaque microbial species composition is related to tooth location and oxygen availability, which may be influenced by toothbrushing or other dental hygiene measures. Our results support that dental plaque, in contrast to the stool microbiome, maintains an inherent stability against ecological perturbations in the oral environment.

HAYSTAC: A Bayesian framework for robust and rapid species identification in high-throughput sequencing data.

Identification of specific species in metagenomic samples is critical for several key applications, yet many tools available require large computational power and are often prone to false positive identifications. Here we describe High-AccuracY and Scalable Taxonomic Assignment of MetagenomiC data (HAYSTAC), which can estimate the probability that a specific taxon is present in a metagenome. HAYSTAC provides a user-friendly tool to construct databases, based on publicly available genomes, that are used for competitive read mapping. It then uses a novel Bayesian framework to infer the abundance and statistical support for each species identification and provide per-read species classification. Unlike other methods, HAYSTAC is specifically designed to efficiently handle both ancient and modern DNA data, as well as incomplete reference databases, making it possible to run highly accurate hypothesis-driven analyses (i.e., assessing the presence of a specific species) on variably sized reference databases while dramatically improving processing speeds. We tested the performance and accuracy of HAYSTAC using simulated Illumina libraries, both with and without ancient DNA damage, and compared the results to other currently available methods (i.e., Kraken2/Bracken, KrakenUniq, MALT/HOPS, and Sigma). HAYSTAC identified fewer false positives than both Kraken2/Bracken, KrakenUniq and MALT in all simulations, and fewer than Sigma in simulations of ancient data. It uses less memory than Kraken2/Bracken, KrakenUniq as well as MALT both during database construction and sample analysis. Lastly, we used HAYSTAC to search for specific pathogens in two published ancient metagenomic datasets, demonstrating how it can be applied to empirical datasets. HAYSTAC is available from https://github.com/antonisdim/HAYSTAC.

Relief food subsistence revealed by microparticle and proteomic analyses of dental calculus from victims of the Great Irish Famine.

Food and diet were class markers in 19th-century Ireland, which became evident as nearly 1 million people, primarily the poor and destitute, died as a consequence of the notorious Great Famine of 1845 to 1852. Famine took hold after a blight (Phytophthora infestans) destroyed virtually the only means of subsistence-the potato crop-for a significant proportion of the population. This study seeks to elucidate the variability of diet in mid-19th-century Ireland through microparticle and proteomic analysis of human dental calculus samples (n = 42) from victims of the famine. The samples derive from remains of people who died between August 1847 and March 1851 while receiving poor relief as inmates in the union workhouse in the city of Kilkenny (52°39' N, -7°15' W). The results corroborate the historical accounts of food provisions before and during the famine, with evidence of corn (maize), potato, and cereal starch granules from the microparticle analysis and milk protein from the proteomic analysis. Unexpectedly, there is also evidence of egg protein-a food source generally reserved only for export and the better-off social classes-which highlights the variability of the prefamine experience for those who died. Through historical contextualization, this study shows how the notoriously monotonous potato diet of the poor was opportunistically supplemented by other foodstuffs. While the Great Irish Famine was one of the worst subsistence crises in history, it was foremost a social disaster induced by the lack of access to food and not the lack of food availability.

Bronze Age population dynamics and the rise of dairy pastoralism on the eastern Eurasian steppe.

Recent paleogenomic studies have shown that migrations of Western steppe herders (WSH) beginning in the Eneolithic (ca. 3300-2700 BCE) profoundly transformed the genes and cultures of Europe and central Asia. Compared with Europe, however, the eastern extent of this WSH expansion is not well defined. Here we present genomic and proteomic data from 22 directly dated Late Bronze Age burials putatively associated with early pastoralism in northern Mongolia (ca. 1380-975 BCE). Genome-wide analysis reveals that they are largely descended from a population represented by Early Bronze Age hunter-gatherers in the Baikal region, with only a limited contribution (∼7%) of WSH ancestry. At the same time, however, mass spectrometry analysis of dental calculus provides direct protein evidence of bovine, sheep, and goat milk consumption in seven of nine individuals. No individuals showed molecular evidence of lactase persistence, and only one individual exhibited evidence of >10% WSH ancestry, despite the presence of WSH populations in the nearby Altai-Sayan region for more than a millennium. Unlike the spread of Neolithic farming in Europe and the expansion of Bronze Age pastoralism on the Western steppe, our results indicate that ruminant dairy pastoralism was adopted on the Eastern steppe by local hunter-gatherers through a process of cultural transmission and minimal genetic exchange with outside groups.

A Robust Framework for Microbial Archaeology.

Microbial archaeology is flourishing in the era of high-throughput sequencing, revealing the agents behind devastating historical plagues, identifying the cryptic movements of pathogens in prehistory, and reconstructing the ancestral microbiota of humans. Here, we introduce the fundamental concepts and theoretical framework of the discipline, then discuss applied methodologies for pathogen identification and microbiome characterization from archaeological samples. We give special attention to the process of identifying, validating, and authenticating ancient microbes using high-throughput DNA sequencing data. Finally, we outline standards and precautions to guide future research in the field.

Comparison of extraction methods for recovering ancient microbial DNA from paleofeces.

OBJECTIVES: Paleofeces are valuable to archeologists and evolutionary biologists for their potential to yield health, dietary, and host information. As a rich source of preserved biomolecules from host-associated microorganisms, they can also provide insights into the recent evolution and changing ecology of the gut microbiome. However, there is currently no standard method for DNA extraction from paleofeces, which combine the dual challenges of complex biological composition and degraded DNA. Due to the scarcity and relatively poor preservation of paleofeces when compared with other archeological remains, it is important to use efficient methods that maximize ancient DNA (aDNA) recovery while also minimizing downstream taxonomic biases. METHODS: In this study, we use shotgun metagenomics to systematically compare the performance of five DNA extraction methods on a set of well-preserved human and dog paleofeces from Mexico (~1,300 BP). RESULTS: Our results show that all tested DNA extraction methods yield a consistent microbial taxonomic profile, but that methods optimized for ancient samples recover significantly more DNA. CONCLUSIONS: These results show promise for future studies that seek to explore the evolution of the human gut microbiome by comparing aDNA data with those generated in modern studies.

The efficacy of whole human genome capture on ancient dental calculus and dentin.

OBJECTIVES: Dental calculus is among the richest known sources of ancient DNA in the archaeological record. Although most DNA within calculus is microbial, it has been shown to contain sufficient human DNA for the targeted retrieval of whole mitochondrial genomes. Here, we explore whether calculus is also a viable substrate for whole human genome recovery using targeted enrichment techniques. MATERIALS AND METHODS: Total DNA extracted from 24 paired archaeological human dentin and calculus samples was subjected to whole human genome enrichment using in-solution hybridization capture and high-throughput sequencing. RESULTS: Total DNA from calculus exceeded that of dentin in all cases, and although the proportion of human DNA was generally lower in calculus, the absolute human DNA content of calculus and dentin was not significantly different. Whole genome enrichment resulted in up to four-fold enrichment of the human endogenous DNA content for both dentin and dental calculus libraries, albeit with some loss in complexity. Recovering more on-target reads for the same sequencing effort generally improved the quality of downstream analyses, such as sex and ancestry estimation. For nonhuman DNA, comparison of phylum-level microbial community structure revealed few differences between precapture and postcapture libraries, indicating that off-target sequences in human genome-enriched calculus libraries may still be useful for oral microbiome reconstruction. DISCUSSION: While ancient human dental calculus does contain endogenous human DNA sequences, their relative proportion is low when compared with other skeletal tissues. Whole genome enrichment can help increase the proportion of recovered human reads, but in this instance enrichment efficiency was relatively low when compared with other forms of capture. We conclude that further optimization is necessary before the method can be routinely applied to archaeological samples.

Long-term genetic stability and a high-altitude East Asian origin for the peoples of the high valleys of the Himalayan arc.

The high-altitude transverse valleys [>3,000 m above sea level (masl)] of the Himalayan arc from Arunachal Pradesh to Ladahk were among the last habitable places permanently colonized by prehistoric humans due to the challenges of resource scarcity, cold stress, and hypoxia. The modern populations of these valleys, who share cultural and linguistic affinities with peoples found today on the Tibetan plateau, are commonly assumed to be the descendants of the earliest inhabitants of the Himalayan arc. However, this assumption has been challenged by archaeological and osteological evidence suggesting that these valleys may have been originally populated from areas other than the Tibetan plateau, including those at low elevation. To investigate the peopling and early population history of this dynamic high-altitude contact zone, we sequenced the genomes (0.04×-7.25×, mean 2.16×) and mitochondrial genomes (20.8×-1,311.0×, mean 482.1×) of eight individuals dating to three periods with distinct material culture in the Annapurna Conservation Area (ACA) of Nepal, spanning 3,150-1,250 y before present (yBP). We demonstrate that the region is characterized by long-term stability of the population genetic make-up despite marked changes in material culture. The ancient genomes, uniparental haplotypes, and high-altitude adaptive alleles suggest a high-altitude East Asian origin for prehistoric Himalayan populations.